AMPA-DART silencing of horizontal cells in mouse retinal slices

Abstract

Purpose : Investigations seeking to determine the function of horizontal cells (HCs) in retinal visual processing often rely on selectively silencing the cells in a subtractive strategy for analytic clarity. HC ablation with DTR, genetic manipulation to knock-out AMPARs, and PSAM-GlyR expression to voltage block the cells have been used with varying degrees of success, but each technique presents compromises. In this presentation, we analyze the selective manipulation of HCs with YM90K-DART, a DNQX-like antagonist of AMPARs that acts exclusively on HaloTagTM expressing cells. We compare the effectiveness of the DART approach on HaloTagTM expressing HCs under different experimental protocols.

Methods : Responses to glutamate in the absence and following washout of YM90K-DART were recorded in dTomato-positive (HaloTagTM expressing) HCs and in cone photoreceptors (PRs) in retinal slices, as well as in enzymatically treated isolated HCs. Selective expression of AAV7m8-CAG-DIO-HaloTagTM-2A-dTomato-WPRE in HCs was achieved with intraocular injection. The HaloTagTM system expresses specific cell surface binding sites, which permanently tether exogenous YM90K-DART, an AMPAR antagonist, to the cell surface to block AMPARs on the targeted cell.

Results : Robust co-labeling in HCs was seen with dTomato fluorescence, and HaloTagTM and Calbindin immunostaining of horizontal cells. Persistent YM90K-DART block (1µM) of AMPARs in HaloTagTM-expressing HCs was seen during 100 µM glutamate application after YM90K-DART superfusion and washout. Disinhibition of Ca_v channels in cones after YM90K-DART treatment of slices with HCs expressing HaloTagTM was seen, with activation curves revealing negative Ca_v channel activation V_½ shifts. When YM90K-DART was applied to enzymatically-isolated HCs, no AMPAR current block was observed.

Conclusions : These results show that YM90K-DART is effective in silencing targeted HCs by selectively blocking their AMPARs. However, with enzymatically treated cells, YM90K-DART may not bind due to enzyme digestion of the HaloTagTM sites.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.